Tuberculosis (TB) has a massive impact on global health. The World Health Organization estimates that one- third of all people worldwide harbor latent TB infections, with sixteen million cases of active disease and nearly two million deaths caused by Mycobacterium tuberculosis (MTB) each year. TB infections are heterogeneous, and current medicines do not work equally well on all MTB bacteria. Efforts to replenish the TB treatment arsenal lag far behind the growing need, particularly for patients with multi- (MDR) or extensively- (XDR) drug-resistant-TB. In bacteria, many critical responses to environmental cues are performed by protein pairs comprised of a sensor kinase that auto-phosphorylates on a histidine and then transfers this phosphate to an aspartate on its cognate response regulator protein. The MTB histidine-aspartate phosphorelay systems are potentially attractive drug targets for two major reasons: First, of the two-component systems in MTB, at least one, MtrAB, is essential for viability even in rich media while others play key roles in virulence or persistence in vivo. Second, the high conservation among phosphoacceptor sites on the response regulators, and the absence of such proteins in mammals, suggest that developing pan-specific and non-toxic inhibitors may be possible. Our strategy is to focus here on the one essential 2CR of MTB, MtrAB, and target the signaling to the response regulator rather than within the kinase, overcoming problems encountered in approaches with 2CRs of other bacteria. In addition to target validation, our work is anticipated to produce at least one chemical scaffold suitable for expansion into a preclinical lead compound. Future plans will expand the targets to the larger family of mycobacterial 2CR response regulators. Specifically in this proof-of-concept work we plan to: Specific Aim 1. Develop an assay for inhibitors of the essential MTB 2CR MtrAB, and format it for HTS. We plan to adapt an innovative in vitro phospho-transfer assay, synthesize a panel of candidate reporter substrates, and evaluate them. One reporter substrate will be selected to conduct Specific Aim 2. Specific Aim 2. Execute an MtrAB phospho-transfer high-throughput screen (HTS). Using the selected synthetic reporter substrate we will conduct an HTS on a 100,000 compound library comprised of compounds obtained from both the Sequella collection and from McMaster University's HTS facility. An anticipated hit rate of ca 0.1 -0.2 % is anticipated, thus providing 100-200 novel structures for Specific Aim 3. 3. Prioritize and expand hit scaffolds from Specific Aim 2. We will select the most promising structures and run in vitro secondary screens. A limited medicinal chemistry expansion will then produce 2-3 distinct scaffolds suitable for pursuit in a subsequent research phase. PUBLIC HEALTH RELEVANCE: Tuberculosis (TB) has a massive impact on global health, and efforts to replenish the TB treatment arsenal lag far behind the growing need, particularly for patients with multi- (MDR) or extensively- (XDR) drug-resistant-TB. We have chosen a promising new drug target, and propose to develop a screening method to identify molecules with significant potential as new drug candidates for the treatment of TB, MDR-TB, and XDR-TB.